Viral and therapeutic control of IFN-beta promoter stimulator 1 during hepatitis C virus infection

Abstract

Viral signaling through retinoic acid-inducible gene-I (RIG-I) and its adaptor protein, IFN promoter-stimulator 1 (IPS-1), activates IFN regulatory factor-3 (IRF-3) and the host IFN-alpha/beta response that limits virus infection. The hepatitis C virus (HCV) NS3/4A protease cleaves IPS-1 to block RIG-I signaling, but how this regulation controls the host response to HCV is not known. Moreover, endogenous IPS-1 cleavage has not been demonstrated in the context of HCV infection in vitro or in vivo. Here, we show that HCV infection transiently induces RIG-I- and IPS-1-dependent IRF-3 activation. This host response limits HCV production and constrains cellular permissiveness to infection. However, HCV disrupts this response early in infection by NS3/4A cleavage of IPS-1 at C508, releasing IPS-1 from the mitochondrial membrane. Cleavage results in subcellular redistribution of IPS-1 and loss of interaction with RIG-I, thereby preventing downstream activation of IRF-3 and IFN-beta induction. Liver tissues from chronically infected patients similarly demonstrate subcellular redistribution of IPS-1 in infected hepatocytes and IPS-1 cleavage associated with a lack of ISG15 expression and conjugation of target proteins in vivo. Importantly, small-molecule inhibitors of NS3/4A prevent cleavage and restore RIG-I signaling of IFN-beta induction. Our results suggest a dynamic model in which early activation of IRF-3 and induction of antiviral genes are reversed by IPS-1 proteolysis and abrogation of RIG-I signaling as NS3/4A accumulates in newly infected cells. HCV protease inhibitors effectively prevent IPS-1 proteolysis, suggesting they may be capable of restoring this innate host response in clinical practice.

Fig. 1.

NS3/4A cleaves IPS-1 to control the host response during HCV infection. (A) Huh7 cells were mock-infected or infected with JFH-1 HCV 2a at a multiplicity of infection of 3. At the indicated h after infection, cells were immunostained with HCV 2a patient serum (green) and anti-IRF-3 serum (red). Nuclei were visualized by staining the cells with DAPI (blue). Cells with nuclear IRF-3 (indicated by arrows) were counted and expressed as a percentage of the total cells in a field. The top left of A shows IRF-3 in Sendai virus-infected control cells. (B) Focus-forming units per ml of HCV within supernatant collected from cultures of Huh7 cells (white bars) or Huh7.5 cells (black bars) at 24 and 48 h after infection with JFH-1 virus. (C) Immunoblot of endogenous IPS-1, NS3, NS5A, and GAPDH in Huh7 cells that were mock-infected (lanes 1–4) or infected with the JFH-1 virus (lanes 5–8) for the time shown above each lane. Arrows mark the positions of full-length (FL) and cleaved (C) forms of IPS-1. (D) Immunoblot of endogenous IPS-1, NS3, and GAPDH in extracts from Huh7 (lane 1), Huh7-K2040 (lane 2), Huh7-HP (lane 3), and Huh7-JFHR (lane 4) cells and Huh7 cells that were mock-infected or infected with the JFH-1 virus (multiplicity of infection = 1) for 96 h (lanes 5 and 6, respectively). (E) Huh7 cells were cotransfected with constructs encoding the Myc-vector (−) or Myc-IPS-1 (+) and either the Flag-vector (lanes 1–2) or a Flag-tagged expression construct encoding WT NS3/4A protease from the HCV 1b Con1 clone (lanes 3–4), protease-deficient Con1 mutant (lanes 5–6), patient isolates (HCV 1a, lanes 7–8; HCV 1b, lanes 9–10; HCV 2b, lanes 11–12), or the JFH-1 clone (lanes 13–14). Expression of Myc-IPS-1, Flag-NS3, ISG56, and GAPDH is shown.

Fig. 2.

NS3/4A cleavage of IPS-1 from the mitochondria is prevented by HCV protease inhibitors. (A) UNS3/4A cells, cultured to suppress (−) or induce (+) NS3/4A expression, were transfected with constructs encoding Flag-vector, WT Flag-IPS-1, or Flag-IPS-1 C508Y mutant. Expression of the Flag-IPS-1 constructs, NS3, and GAPDH is shown. (B Upper) aa sequence comparison of the NS3/4A cleavage sites (denoted by the arrow) of IPS-1 and the NS5A/5B junction in the HCV 1b polyprotein. Cleavage site positions and IPS-1 aa numbers are shown. Identical and conserved aa residues are boxed and underlined, respectively. (B Lower) Structural representation of IPS-1 denoting the aa positions of the CARD, Proline-rich region (Pro), transmembrane domain (TM), and the NS3/4A cleavage site at C508. (C) Huh7 cells that were mock-infected or infected with HCV for 48 h were immunostained for endogenous IPS-1 and HCV proteins. Nuclei were stained with DAPI. For the HCV-infected cells, the brightness of the IPS-1 panels was increased to 200% to allow visualization of the redistributed IPS-1. (Magnification: ×64.) (D) Immunoblot analysis of IPS-1 within mitochondria preparations. (Left) Mitochondria recovered from Huh7 cells (control, lane 1) or cells harboring the replicating full-length HCV-N genome (20) (lanes 2 and 4) and their IFN-cured counterparts (lanes 3 and 5). (Right) Mitochondria preparations from Huh7 cells were analyzed alone (lane 1) or after incubating with buffer (lanes 2–3), MB-scNS3 (lanes 4–5), or MB-scNS3 and 10 μM SCH6 (lanes 6–7). The mixtures in lanes 2–7 were further separated into soluble (S) and pellet (P) fractions before immunoblot analysis. Arrows denote the positions of IPS-1, which is recovered as multiple products from mitochondria preparations (8). The lower panels show the abundance of the mitochondrial membrane-associated 39-kDa subunit of cytochrome C oxidase I (CI 39) (21). (E) Huh7, Huh7-A7, or Huh7-HP cells were treated with DMSO (lanes 1–3), 10 μM SCH6 (lanes 4–6), or 1 μM ITMN-C (lanes 7–9) for 48 h before immunoblot analysis of endogenous IPS-1, NS3, and GAPDH.

Fig. 3.

Cleavage of IPS-1 blocks RIG-I interaction and disrupts signaling to IFN-β, but signaling is restored by protease inhibitor treatment. (A) UNS3/4A cells, cultured to suppress (−) or induce (+) NS3/4A expression, were cotransfected with constructs encoding Myc-N-RIG and Flag-vector (lanes 1–2), Flag-IPS-1 (lanes 3–4), Flag-IPS-1 C508Y (lanes 5–6), or Flag-IPS-1 1-508 (lanes 7–8). Extracts were subject to immunoprecipitation by using anti-Myc antibody and analyzed by immunoblotting for the presence of the WT or mutant Flag-IPS-1 protein and Myc-N-RIG (upper panels). The lower panels show the abundance of NS3, Flag-IPS-1, and GAPDH in the input extracts. (B and C) UNS3/4A cells, cultured to suppress (black bars) or induce (white bars) NS3/4A expression, were transfected with Renilla luciferase and IFN-β-luciferase plasmid constructs and a plasmid encoding the Flag-vector (V), Flag-IPS-1 (IPS), Flag-IPS-1 1-508 (1-508), Flag-IPS-1 C508Y (C508Y), Flag-N-RIG (RIG), or IRF-3–5D (5D). Cells were incubated for 24 h in medium alone (B) or medium with DMSO or 1 μM ITMN-C (C), and extracts were subjected to the dual luciferase assay. Bars show relative IFN-β-luciferase activity (±SD).

Fig. 4.

In vivo redistribution and cleavage of IPS-1 during chronic HCV infection. Confocal micrographs of a 0.3-μm optical section of immunostained sections from normal donor liver (A) or liver biopsy from a patient with chronic HCV infection (B). (Magnification: ×40.) Shown are individual and merged images of sections stained with DAPI and antiserum specific to either IPS-1 (red) and NS3 (green) (Left) or IPS-1 (red) and COX-I (green) (Right). (B Lower) Images from the same tissue specimen shown in B Upper and were collected at a depth of 9 μm within the 32-μm-thick specimen. (B Lower Left) Images correspond to boxed area 1 (shown in the lower right corner of B Upper Left) of hepatocytes that stained negative for NS3. (B Lower Right) Images correspond to boxed area 2 (shown in the lower right corner of B Upper Left) of an adjacent focal area of hepatocytes that stained positive for NS3. (C) Immunoblot of protein extracts from Huh7 and Huh7-HP cells (lanes 1–2, respectively), specimens from three unused, normal donor livers (lanes 3–5), or specimens of liver from different patients chronically infected with HCV (lanes 6–9). Panels show the abundance of IPS-1, ISG15, or GAPDH. Arrows point to the positions of IPS-1 and native ISG15. The vertical line denotes ISG15 conjugation products; asterisks denote nonspecific bands that are not reproducibly observed.